Kenysha Y.J. Clear, Adam S. Wilson, Tiffany M. Newman, David R. Soto-Pantoja, Carol A. - PowerPoint Presentation

1. Kenysha Y.J. Clear, Adam S. Wilson, Tiffany M. Newman, David R. Soto-Pantoja, Carol A. Shively, and Katherine L. CookHypertension and Vascular Research, Wake Forest School of Medicine 575 Patterson Ave, Winston-Salem, NC 27101Investigating lactate isoforms as a potential mechanism to reduce obesity –mediated breast cancer riskAbstractThe production of lactate is often associated with cellular stress and as a metabolic waste product. Under normal, non-stressful conditions, the metabolism of glucose via glycolysis results in an overarching production of pyruvate, which can then be used in the mitochondria in the tricarboxylic acid cycle (TCA) to generate adenosine triphosphate (ATP) for cellular processes and function. In a stressful environment, like that lacking oxygen and/or uncontrolled proliferation, there is a shift in the glycolytic pathway to produce more lactate from pyruvate thereby resulting in less mitochondrial utilization to produce ATP. Lactate can also be used as an alternative energy source by being converted back into pyruvate or can also be shuttled out of the cell by the monocarboxylic transporters (MCT) 1 and MCT4 to promote immunosuppression within the microenvironment. Obesity is a prevalent health condition in the United States and is a modifiable risk factor for breast cancer development in postmenopausal women. As poor diet is a major contributing factor for the development of obesity and subsequent breast cancer, it is essential to maintain a healthy dietary lifestyle as a way to prevent breast cancer development. Balanced diets can also maintain essential intestinal and gut health by maintaining the symbiotic relationships between the microbes found there, but may also play a role on microbes found in distal organs and tissues, like the breast. Using a nonhuman primate (NHP) model, our lab performed metabolomics on mammary glands from M. fascularis NHP fed a Western or Mediterranean diet for 2.5 years. With the animals separated in groups based on body fat composition, we were able to look at the impact of adiposity within dietary patterns on mammary gland (MG) metabolism. Comparing metabolites identified from these MGs, NHP with the highest body fat composition displayed decreased lactate concentrations when compared with lean NHP breast tissue levels. In addition to the increased levels of lactate found in these MGs independent of diet, elevated proportional abundance of lactate-producing bacteria were present in lean NHP breast tissue, suggesting a potential role of the microbiome in the regulation of gland-specific lactate. C57BL/6 mice demonstrate susceptibility to dietary induced obesity. When analyzing gene expression and protein levels of lactate dehydrogenase (LDH) enzyme isoforms responsible for converting pyruvate to lactate, LDHA (L-lactate isoform specific) and LDHD (D-lactate isoform specific), LDHA and LDHD were higher in MG tissue of low-fat Control diet-fed mice compared to Western diet-fed mice. These data suggest the regulation of lactate metabolism by obesity within the mammary gland compartment may be mediated through repression of enzyme activity. Future goals of the study include to investigate the impact of L-lactate and D-lactate isoforms on obesity-mediated breast cancer risk, breast metabolic pathways, and localized gland inflammation. Moreover, we plan to explore the effect of the breast microbiome on regulating localized lactate levels within the MG to potentially modify breast cancer risk. BackgroundLactate can mediate DNA repairLactate can suppress inflammationLactate can be an alternative energy sourceCan lactate reduce breast cancer risk? Figure 1: NHP subjects consuming Western or Mediterranean diet categorized by body fat composition display differential metabolite profiles. A. NHP body mass index (BMI) by diet and tertile. B. Body fat composition as determined by DEXA scan shown by diet and body weight tertile. Body composition correlated with NHP BMI. C. Principal component analysis plot of MG metabolites by diet and tertile. MG metabolomics profiles by body weight tertile in Western diet (but not Mediterranean diet) fed animals significantly differ. Relative abundance of retinol (D), lactate (E), or maltose (F). n=6-7; *p<0.05. Bar and whisker plot represents min to max distribution with the median value indicated by mid-line, individual NHP shown as single plot, and mean value listed above/below bar whisker plot.Lean NHP subjects display elevated breast lactate concentrations. Figure 3: Mice consuming a Western diet have reduced mammary gland lactate dehydrogenase-A protein levels when compared with low fat Control diet-fed animals. *p<0.05 Decreased lactate production in mammary tissue modulated by adiposity may be result of decreased host LDHA enzyme levels. MethodsUntargeted metabolomics was performed on snap-frozen breast tissue from a non-human primate cohort fed a Western or Mediterranean diet for 2.5 years. Results were aggregated by subject adiposity as determined metabolites regulated by obesity in the breast tissue.16S sequencing was performed on DNA isolated from NHP breast tissue. 16S sequencing (V4 region) was performed on an Illumina to determine bacteria populations in samples. Raw Fastq files were quality-filtered and clustered into 97% similarity operational taxonomic units (OTUs) using the mothur software package. High quality reads will be classified using Greengenes as the reference database. We obtain a consensus taxonomy for each OTU. We then aggregate OTU abundances into taxonomies, and plot the relative abundances of the most abundant ones. Results were grouped by subject adiposity and breast microbiota populations regulated by obesity was explored.Female C57BL/6 mice were fed a low fat Control or Western diet for 10 weeks. Mammary gland tissue was excised and snap-frozen. Protein lysates from mammary gland tissue were used for Western blot hybridization to determine diet effects on mammary gland lactate dehydrogenase.MCF7 breast cancer cells were plated in 2% serum containing DMEM media in an ACEA xCelligence cell analyzer. Cells were treated with increasing doses of L-lactate or D-lactate. Cell index was measured by electrical impedance every 6 hours for 48 hours to determine the effects of lactate isoforms on breast cancer cell viability. Conclusions Heavier NHP subjects, regardless of what diet they were consuming, displayed reduced lactate and maltose metabolite concentrations in their breast tissue. Both maltose and lactate can be generated by bacterial metabolic processes and may be generated by localized microbiome populations.16S sequencing of NHP breast tissue indicates lean subjects on either diet display elevated lactate-producing bacterial populations.Western diet consuming female mice display reduced lactate dehydrogenase-A protein levels in their mammary tissue, suggesting adiposity regulation of localized lactate may be a host driven process.Treatment with D-lactate, a bacterial derived isoform, decreased MCF7 cell viability.D-lactate produced by localized probiotic bacteria in the breast may represent a novel mechanism for breast cancer prevention. Acknowledgements: This work was supported by an American Cancer Society Research Scholar Grant RSG-16-204-01-NEC (KLC), a Susan G. Komen Career Catalyst grant CCR18547795 (KLC), and the Department of Defense Breast Cancer Research Program Breakthrough Level 2 Award (KLC)Figure 2: Lean NHP regardless of diet contain higher abundance of lactate-producing bacteria. Lean Western diet-fed NHP display elevated Enterocooccaceae, Lactobacillus, Lactococcus, and Lachnospiraceae than heavy Western diet-fed NHPs. Lean Mediterranean diet-fed NHP display elevated breast Lactobacillus compared to populations of NHP with higher adiposity on the same diet as identified by 16S. n=5-6; *p<0.05. Lean NHP subjects contain increased abundance of lactate-producing bacteria.Figure 4: MCF7 ER+ breast cancer cells treated with D-lactate but not L-lactate display reduced cell viability. *p<0.05 Bacterial derived D-lactate reduces breast cancer cell viability.LDHAControl1 uM L-lactate10 uM L-lactate100 uM L-lactate1 mM L-lactateControl1 uM D-lactate10 uM D-lactate100 uM D-lactate1 mM D-lactate